The p53 tumor suppressor gene is a major target for inactivation in human cancer. In non-stressed normal cells, the p53 protein is practically latent, owing to its being present in very low amounts and in a rather inactive molecular form. The relative lack of activity of p53 under such conditions is largely due to the action of Mdm2, which targets p53 for rapid degradation and, furthermore, maintains p53 in a complex that is incompatible with efficient performance of p53 as a transcription factor. In response to a variety of stress signals, particularly those that are closely linked with cancer (e.g. genomic damage, oncogene activation), p53 is freed of the inhibitory effects of Mdm2. This results in stabilization and activation of p53, leading to a multitude of biochemical and biological effects. The consequence of such p53 activation, when occurring in an incipient cancer cell, is the elimination of such cell from the replicative pool, preventing its further progression into a life-threatening tumor. The long term objective of this project is to elucidate the mechanisms responsible for maintaining p53 inactive in non-stressed cells, the biochemical events that enable p53 activation in response to cancer-related stress, and the molecular basis for the failure of p53 to function properly in human cancer cells, even when retained in its wild type form. Specifically, this work will address the impact of covalent modifications on Mdm2, particularly acetylation, evaluate the exact nature of the changes that are imposed on p53 as a consequence of Mdm2 activity, investigate the idea that some of the inhibitory effects of Mdm2 may involve its interaction with the chromatin of p53 target genes in a manner leading to transcriptional silencing, explore the possibility that dominant negative forms of p63, a p53 family member, are responsible for the failure of p53 to function as an effective tumor suppressor in some human cancers, and elucidate the regulation of p53 activation by cell-cell interaction and its relevance for the conversion of epithelial tumors to a more malignant state. The methodology will include a combination of cell-free assays for protein activity, mutational analysis and genetic manipulation of cultured cells, analysis of protein-protein interactions, gene expression analysis, and in vivo experiments in normal and cancer-bearing mice. Success of the proposed experiments should allow a better understanding of the regulation of p53 activity and its subversion in cancer, and provide clues for improved cancer therapy.